Design and Synthesis of Collybolide Probes for Kappa-Opioid Receptor Abstract: While the mu-opiate analgesics such as morphine are effective for severe pain, their use is limited due to unwanted side effects such as respiratory depression, physical dependence, psychological addiction, abuse liability and itching. Studies suggest that selective kappa-opioid receptor (KOR) agonists biased towards G- protein signaling could be novel therapeutics to treat pain with reduced side effects. Deciphering the structural requirements essential for KOR selectivity through novel chemical probes is the first step in developing selective- and biased-KOR ligands. In the quest for new KOR chemical probes, we identified collybolide, a non-nitrogenous sesquiterpene natural product extracted from the mushroom Collybia maculata as a highly selective KOR agonist having in vivo analgesic and antipruritic activity. Collybolide has a furyl-?-lactone core similar to that of salvinorin A, however differs from the latter in exhibiting functional selectivity along with a 50- fold higher potency in blocking non-histamine-mediated itch. The long-term goal of this research program is to develop KOR based analgesics and anti-pruritic drugs with reduced side effects. The objective of this project is the development of novel collybolide probes for identifying selective- and biased-KOR ligands with desirable pharmacological profiles for use in vivo. Our hypothesis is that rational structural diversification of collybolide will provide novel probes to gain important structural information that could lead to the development of effective therapeutics for the treatment of pruritus and pain with reduced side effects. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following specific aims: 1) Synthesis and evaluation of first- generation collybolide probes for identifying selective-and biased KOR ligands; 2) Design, synthesis and in vivo evaluation of optimized collybolide analogues with drug-like properties to identify potential analgesics and anti-pruritus agents with reduced side-effects. The proposed research will be performed by a highly qualified team of researchers having synergistic expertise in natural products isolation, organic synthesis, and medicinal chemistry as well as in pharmacology, neuroscience, computational docking, and protein crystallization. The studies in this application will comprehensively examine the structure?activity relationships of collybolide and are expected to yield a set of selective- and biased-?OR ligands with the potential of becoming lead drug candidates for itch and pain with reduced side effects. Further, high-throughput screening at the NIMH-PDSP against 50 CNS receptors could find new hits, which could be optimized further to explore new therapeutic opportunities.